A cell-free system for reconstitution of transport between prevacuolar compartments and vacuoles in Saccharomyces cerevisiae.
A cell-free system for reconstitution of transport between prevacuolar compartments and vacuoles in Saccharomyces cerevisiae.
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用于重建酿酒酵母液泡前区室和液泡之间运输的无细胞系统。
DOI：
10.1007/978-1-59745-261-8_4
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Vida,ThomasA
中科院分区：
文献类型：
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作者：
Vida,ThomasA
遗传学方法已经揭示了50多个基因参与传递可溶性酶原如羧肽酶Y（CPY）的溶酶体样空泡在酿酒酵母。这些基因中至少有20个在前液泡内体样区室（PVC）和液泡之间的运输中起作用。为了获得这些功能的生物化学途径，作者开发了一种无细胞测定法，该测定法测量可溶性酶原体外的转运偶联蛋白水解成熟。脉冲追踪放射性标记后，使用具有规定孔径的聚碳酸酯过滤器裂解酵母原生质球。差速离心富集125，000g膜沉淀中含有proCPY（p2CPY）的PVC，并用作供体膜。非放射性标记的原生质球也用聚碳酸酯过滤器裂解，但收集富含液泡的15，000g膜沉淀并用作受体膜。当这两个粗膜小球一起孵育与腺苷三磷酸和胞质蛋白提取物，近50%的放射性标记的p2CPY可以被加工成成熟的空泡形式，mCPY。这种无细胞系统允许与VPS基因产物功能偶联的室间转运的重建。
Genetic approaches have revealed more than 50 genes involved in the delivery of soluble zymogens like carboxypeptidase Y (CPY) to the lysosome-like vacuole inSaccharomyces cerevisiae. At least 20 of these genes function in transport between the prevacuolar endosome-like compartment (PVC) and the vacuole. To gain biochemical access to these functions, the authors developed a cell-free assay that measures transport-coupled proteolytic maturation of soluble zymogensin vitro. A polycarbonate filter with a defined pore size is used to lyse yeast spheroplasts after pulse-chase radiolabeling. Differential centrifugation enriches for PVCs containing proCPY (p2CPY) in a 125,000gmembrane pellet and is used asdonormembranes. Nonradiolabeled spheroplasts are also lysed with a polycarbonate filter but a 15,000gmembrane pellet enriched for vacuoles is collected and used asacceptormembranes. When these two crude membrane pellets are incubated together with adenosine triphosphate and cytosolic protein extracts, nearly 50% of the radiolabeled p2CPY can be processed to the mature vacuolar form, mCPY. This cell-free system allows reconstitution of intercompartmental transport coupled to the function ofVPSgene products.